1. Field of the Invention
This invention relates to radiotherapeutic agents and peptides, radiodiagnostic agents and peptides, and methods for producing such labeled radiodiagnostic and radiotherapeutic agents. Specifically, the invention relates to receptor-binding vasoactive intestinal peptides (including native vasoactive intestinal peptide (VIP) and fragments, derivatives, analogues and mimetics thereof), and embodiments of such compounds labeled with gamma-radiation emitting isotopes such as technetium-99m (Tc-99m), as well as methods and kits for making, radiolabeling and using such peptides to image sites in a mammalian body. The invention also relates to receptor binding vasoactive intestinal peptides and derivatives, analogues and mimetics thereof, labeled with cytotoxic radioisotopes such as rhenium-186 (.sup.188 Re) and rhenium-188 (.sup.188 Re), and methods and kits for making, radiolabeling and using such compounds therapeutically in a mammalian body.
2. Description of the Prior Art
Native vasoactive intestinal peptide (VIP) is a 28 amino acid peptide that was first isolated from hog upper small intestine (Said and Mutt. 1970, Science 169: 1217-1218). This peptide belongs to a family of structurally-related, small peptides that includes helodermin, secretin, the somatostatins, and glucagon. The peptide has the formula: EQU HSDAVFTDNYTRLRKQMAVKKYLNSILN.amide (SEQ. ID NO.:1) Formula I
(where single-letter abbreviations for amino acids can be found in Zubay. Biochemistry 2d ed., 1988. MacMillan Publishing: New York, p. 33).
The biological effects of VIP are mediated by the activation of membrane-bound receptor proteins that are coupled to the intracellular cyclic adenosine monophosphate signalling system. VIP regulates a variety of different biological activities in tissues and organs. It modulates cellular metabolic activities and regulates exocrine and endocrine secretions. It also induces relaxation of smooth muscle and causes vasodilatory effects. VIP is also involved in the regulation of cellular proliferation and survival in a number of different cell types, including keratinocytes, smooth muscle cells, sympathetic neuroblasts, hippocampal cells and, in vitro, NIH 3T3 cells.
VIP receptors are widely distributed throughout the gastrointestinal tract and are also found in various other cell types. Large numbers of VIP receptors are expressed in rumor cells of, for example, adenocarcinomas, breast cancers, melanomas, neuroblastomas and pancreatic carcinomas. In fact, expression of high affinity binding sites for VIP (comprising the VIP receptor protein) is a marker for these tumor cells. Specific binding of VIP to these cells can be exploited as a marker to locate and identify such tumor cells in vivo.
A variety of radionuclides are known to be useful for radioimaging, including .sup.67 Ga, .sup.99m Tc (hereinafter Tc-99m), .sup.111 In, .sup.123 I, .sup.125 I, .sup.169 Yb or .sup.186 Re. A number of factors must be considered for optimal radioimaging in humans. To maximize the efficiency of detection, a radionuclide that emits gamma energy in the 200 to 200 keV range is preferred. To minimize the absorbed radiation dose to the patient, the physical half-life of the radionuclide must be as short as the imaging procedure will allow. To allow for examinations to be performed on any day and at any time of the day, it is advantageous to have a source of the radionuclide always available at the clinical site.
Methods for radioiodinating VIP analogues at tyrosine residues in the VIP sequence (Tyr.sup.10 or Tyr.sup.22) using .sup.123 I or .sup.125 I are known in the prior art. These radioiodinated species have also been used to assess VIP binding to receptors on tumor cells.
Boissard et al., 1986, Cancer Res. 46: 4406-4413 describe radioiodination of VIP and binding to human colon adenocarcinoma cells.
El Battari et al., 1988, J. Biol. Chem. 263: 17685-17689 describe raioiodination of VIP and binding to human colon adenocarcinoma cells.
Shaffer et al., 1987, Peptides 8: 1101-1106 disclose radioiodination of VIP and binding to human small cell and non-small cell carcinoma cells.
Svoboda et al., 1988, Eur. J. Biochem. 176: 707-713 describe radioiodination of VIP and binding to rat transformed pancreatic acinar cells.
Gespach et al., 1988, Cancer Res. 48: 5079-5083 disclose radioiodination of VIP and binding to human breast cancer cells.
Muller et al., 1989, J. Biol. Chem. 264: 3647-3650 disclose radioiodination of VIP and binding to human neuroblastoma cells.
Lee et al., 1990, Peptides 11: 1205-1210 disclose radioiodination of VIP and binding to human small cell and non-small cell carcinoma cells.
Park et al., 1990, Cancer Res. 50: 2773-2780 describe radioiodination of VIP and binding to human gastric cancer cells.
Bellan et al., 1992, Exp. Cell Res. 200: 34-40 disclose radioiodination of VIP and binding to human melanoma cells.
Moody et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4345-4349 disclose radioiodination of VIP and binding to non-small cell lung carcinoma cells.
Virgolini et al., 1994, Cancer Res. 54: 690-700 describe radioiodination of VIP and binding to primary tumors and tumor cell lines.
Methods for radioiodinating VIP analogues at Y.sup.10 or Y.sup.22 using .sup.131 I are known in the prior art.
Hassan et al., 1994, Nucl. Med. Biol. 21: 865-872 disclose radioiodination of VIP and in vivo distribution of radiolabel in a rat by scintigraphy.
These methods have application for enabling detection of tumor cells in vivo by radioimaging, particularly radioscintigraphy. VIP is a useful marker for such radioimaging, because many different tumor cells express a high affinity binding site for VIP (the VIP receptor protein). However, radioiodinated peptides have significant commercial disadvantages. .sup.123 I is both expensive and in limited supply. Also, approved radioiodinated radiopharmaceuticals normally cannot be prepared at the clinical site.
Tc-99m is a preferred radionuclide because it emits gamma radiation at 140 keV, it has a physical half-life of 6 hours, and it is readily available on-site using a molybdenum-99/technetium-99m generator. Other radionuclides used in the prior art are less advantageous than Tc-99m. This can be because the physical half-lives of such radionuclides are longer, resulting in a greater amount of absorbed radiation dose to the patient (e.g., indium-111). Alternatively, the gamma radiation energies of such alternate radionuclides are significantly lower (e.g., iodine-125) or higher (e.g., iodine-131) than Tc-99m and are thereby inappropriate for quality scintigraphic imaging. Furthermore, many disadvantageous radionuclides cannot be produced using an on-site generator.
Tc-99m is a transition metal that is advantageously chelated by a metal complexing moiety. Radiolabel complexing moieties capable of binding Tc-99m can be covalently linked to various specific binding compounds to provide a means for radiolabeling such specific binding compounds. This is because the most commonly available chemical species of Tc-99m, pertechnetate (TcO.sub.4.sup.-), cannot bind directly to most specific binding compounds strongly enough to be useful as a radiopharmaceutical. Complexing of Tc-99m with such radiolabel complexing moieties typically entails chemical reduction of the pertechnetate using a reducing agent such as stannous chloride.
Although Tc-99m is the preferred radionuclide for scintigraphic imaging, it has not been widely used for labeling peptides (see Lamberts, 1991, J. Nucl. Med. 32: 1189-1191). This is because methods known in the prior art for labeling larger protein molecules (i.e., &gt;10,000 daltons in size) with Tc-99m are not suitable for labeling peptides (having a molecular size less than 10,000 daltons). Consequently, it is necessary to radiolabel most peptides by covalently attaching a radionuclide chelating moiety to the peptide, so that the chelator is incorporated site-selectively at a position in the peptide that will not interfere with the specific binding properties of the peptide.
Methods for labeling peptides with Tc-99m are disclosed in co-owned U.S. Pat. No. 5,225,180 and in co-pending U.S. patent application Ser. No. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,811,394; Ser. No. 07/807,062, now U.S. Pat. No. 5,443,815; Ser. No. 07/851,074, now abandoned, a divisional of which issued as U.S. Pat. No. 5,711,931; Ser. No. 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934; Ser. No. 07/886,752, now abandoned, which issued as U.S. Pat. No. 5,849,260; Ser. No. 07/893,981, now U.S. Pat. No. 5,508,020; Ser. No. 07/902,935, now U.S. Pat. No. 5,716,596; Ser. No. 07/955,466, now abandoned; Ser. No. 07/977,628, now U.S. Pat. No. 5,405,597; Ser. No. 08/019,864, now U.S. Pat. No. 5,552,525; Ser. No. 08/044,825, now abandoned, which issued as U.S. Pat. No. 5,645,815; Ser. No. 08/073,577, now U.S. Pat. No. 5,561,220; Ser. Nos. 08/092,355; 08/095,760, now U.S. Pat. No. 5,620,675; and Ser. No. 08/210,822, now abandoned, and PCT International Applications PCT/US92/00757, PCT/US92/10716, PCT/US93/02320, PCT/US93/03687, PCT/US93/04794, PCT/US93/05372, PCT/US93/06029, PCT/US93/09387, and PCT/US94/01894, which are hereby incorporated by reference. However none of these references disclose specifically how to prepare technetium-99m -labeled vasoactive intestinal peptides.
Methods for preparing Tc-99m complexes are known in the art and examples are provided below for general reference:
Byrne et al., U.S. Pat. Nos. 4,434,151, 4,575,556 and 4,571,430 describe homocysteine thiolactone-derived bifunctional chelating agents.
Fritzberg, U.S. Pat. No. 4,444,690 describes a series of technetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.
Nosco et al., U.S. Pat. No. 4,925,650 describe Tc-99m chelating complexes.
Kondo et al., European Patent Application, Publication No. 483704 A1 disclose a process for preparing a Tc-99m complex with a mercapto-Gly-Gly-Gly moiety.
European Patent Application No. 84109831.2 describes bisamido, bisthiol Tc-99m ligands and salts thereof as renal function monitoring agents.
Davison et al., 1981, Inorg. Chem. 20: 1629-1632 disclose oxotechnetium chelate complexes.
Fritzberg et al., 1982, J. Nucl. Med. 23: 592-598 disclose a Tc-99m chelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.
Byrne et al., 1983, J. Nucl. Med. 24: P126 describe homocystine-containing Tc-99m chelating agents.
Bryson et al., 1988, Inorg. Chem. 27: 2154-2161 describe neutral complexes of technetium-99 which are unstable to excess ligand.
Misra et al., 1989, Tet. Lett. 30: 1885-1888 describe bisamine bisthiol compounds for radiolabeling purposes.
The use of chelating agents for radiolabeling specific-binding compounds is known in the art and examples are provided below for general reference:
Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturing and purifying Tc-99m chelate-conjugated monoclonal antibodies.
Stavrianopoulos, U.S. Pat. No. 4,943,523 teach detectable molecules comprising metal chelating moieties.
Fritzberg et al., European Patent Application No. 86100360.6 describe dithiol, diamino, or diamidocarboxylic acid or amine complexes useful for making technetium-labeled imaging agents.
Albert et al., UK Patent Application 8927255.3 disclose radioimaging using somatostatin derivatives such as octreotide labeled with .sup.111 I via a chelating group bound to the amino-terminus.
Albert et al., European Patent Application No. WO 91/01144 disclose radioimaging using radiolabeled peptides related to growth factors, hormones, interferons and cytokines and comprised of a specific recognition peptide covalently linked via an amino group of said peptide to a radionuclide chelating group.
Fischman et al., International Patent Application, Publication No. WO93/13317 disclose chemotactic peptides attached to chelating moieties.
Kwekkeboom et al., 1991, J. Nucl. Med. 32: 981 Abstract #305 relates to radiolabeling somatostatin analogues with .sup.111 In.
Albert et al., 1991, Abstract LM10, 12th American Peptide Symposium: 1991 describe uses for .sup.111 In-labeled diethylene-triaminopentaacetic acid-derivatized somatostatin analogues.
Cox et al., 1991, Abstract. 7th International Symposium on Radiopharmacology, p. 16, disclose the use of, Tc-99m-, .sup.131 I- and .sup.111 In-labeled somatostatin analogues in radiolocalization of endocrine tumors in vivo by scintigraphy.
Methods for labeling certain specific-binding compounds, mainly large proteins, with Tc-99m are known in the prior art, and examples are provided below for general reference:
Hnatowich, U.S. Pat. No. 4,668,503 describe Tc-99m protein radiolabeling.
Tolman, U.S. Pat. No. 4,732,684 describe conjugation of targeting molecules and fragments of metallothionein.
Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional coupling agents useful in forming conjugates with biological molecules such as antibodies.
Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protected mercaptoacetylglycylglycine-based chelators for labeling proteins.
Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabeling of proteins comprised of at least one "pendent" sulfhydryl group.
Fritzberg et al., U.S. Pat. No. 5,091,514 describe various S-protected mercaptoacetylglycylglycine-based chelators for labeling proteins.
Gustavson et al., U.S. Pat. No. 5,112,953 disclose Tc-99m chelating agents for radiolabeling proteins.
Kasina et al., U.S. Pat. No. 5,175,257 describe various combinations of targeting molecules and Tc-99m chelating groups.
Dean et al., U.S. Pat. No. 5,180,816 disclose methods for radiolabeling a protein with Tc-99m via a bifunctional chelating agent.
Sundrehagen, International Patent Application, Publication No. WO85/03231 disclose Tc-99m labeling of proteins.
Reno and Bottino, European Patent Application 87300426.1 disclose radiolabeling antibodies with Tc-99m.
Bremer et al., European Patent Application No. 87118142.6 disclose Tc-99m radiolabeling of antibody molecules.
Pak et al., European Patent Application No. WO 88/07382 disclose a method for labeling antibodies with Tc-99m.
Goedemans et al., PCT Application No. WO 89/07456 describe radiolabeling proteins using cyclic thiol compounds, particularly 2-iminothiolane and derivatives.
Dean et al., International Patent Application, Publication No. WO89/12625 teach bifunctional coupling agents for Tc-99m labeling of proteins.
Schoemaker et al., International Patent Application, Publication No. WO90/06323 disclose chimeric proteins comprising a metal-binding region.
Thornback et al., EPC Application No. 90402206.8 describe preparation and use of radiolabeled proteins or peptides using thiol-containing compounds, particularly 2-iminothiolane.
Gustavson et al., International Patent Application, Publication No. WO91/09876 disclose Tc-99m chelating agents for radiolabeling proteins.
Rhodes, 1974, Sem. Nucl. Med. 4: 281-293 teach the labeling of human serum albumin with technetium-99m.
Khaw et al., 1982, J. Nucl. Med. 23: 1011-1019 disclose methods for labeling biologically active macromolecules with Tc-99m.
Schwartz et al., 1991, Bioconjugate Chem. 2: 333 describe a method for labeling proteins with Tc-99m using a hydrazinonicotinamide group.
Attempts at labeling peptides have been reported in the prior art.
Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides for imaging localized T-lymphocytes.
Morgan et al., U.S. Pat. No. 4,986,979 disclose methods for imaging sites of inflammation.
Flanagan et al., U.S. Pat. No. 5,248,764 describe conjugates between a radiolabel chelating moiety and atrial natiuretic factor-derived peptides.
Ranby et al., 1988, PCT/US88/02276 disclose a method for detecting fibrin deposits in an animal comprising covalently binding a radiolabeled compound to fibrin.
Lees et al., 1989, PCT/US89/01854 teach radiolabeled peptides for arterial imaging.
Morgan et al., International Patent Application, Publication No. WO90/10463 disclose methods for imaging sites of inflammation.
Flanagan et al., European Patent Application No. 90306428.5 disclose Tc-99m labeling of synthetic peptide fragments via a set of organic chelating molecules.
Stuttle, PCT Application, Publication No. WO 90/15818 suggests Tc-99m labeling of RGD-containing oligopeptides.
Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecular recognition units" with "effector domains".
Cox, International Patent Application No. PCT/US92/04559 discloses radiolabeled somatostatin derivatives containing two cysteine residues.
Rhodes et al., International Patent Application, Publication No. WO93/12819 teach peptides comprising metal ion-binding domains.
Lyle et al, International Patent Application, Publication No. WO93/15770 disclose Tc-99m chelators and peptides labeled with Tc-99m.
Coughlin et al, International Patent Application, Publication No. WO93/21151 disclose bifunctional chelating agents comprising thiourea groups for radiolabeling targeting molecules.
Knight et al., 1990, 37th Annual Meeting of the Society of Nuclear Medicine, Abstract #209, claim thrombus imaging using Tc-99m labeled peptides.
Babich et al., 1993, J. Nucl. Med. 34: 1964-1974 describe Tc-99m labeled peptides comprising hydrazinonicotinamide derivatives.
Radiolabeled derivatives of VIP receptor binding peptides and fragments, analogues and mimetics thereof can also be used therapeutically. For these applications, cytotoxic radioisotopes rhenium-186 and rhenium-188 are particularly advantageous.
Thus there remains a need for synthetic (to make routine manufacture practicable and to ease regulatory acceptance) VIP receptor binding peptides. derivatives, analogues and mimetics which can be radiolabeled with Tc-99m, for use as scintigraphic agents and which can be radiolabeled with rhenium-186 or henium-188 for use as radiotherapeutic agents. Small synthetic VIP receptor binding peptides, derivatives, analogues and mimetics thereof which contain chelating groups for chelating Tc-99m, Re-186 or Re-188 and their Tc-99m, Re-186 and Re-188 labeled derivatives are provided by this invention that specifically fulfill this need.